Paracoccidioides brasiliensis is a thermally dimorphic fungus, growing in its yeast form at 37(C and in mycelium form at 23(C. Infection by P. brasiliensis is prevalent among rural farm workers in South America, Central America, and Mexico, commonly affecting the lung, lymphoid, and mucocutaneous tissues. Although the etiology remains unclear, it is assumed that patients are infected by the mycelium form present in soil. In patients exhibiting paracoccidioidomycosis (PCM), only the yeast form is detected. The yeast form is also infectious, particularly when handled in culture. Recently, we described the isolation of glycosylinositol phosphorylceramides from the yeast and mycelium forms of P. brasiliensis, including an acidic antigen (band 1) reactive with the sera of all patients tested exhibiting PCM. A terminal Gal residue in its furanosidic form was found to be immunodominant in the serologic reaction. A second acidic glycosphingolipid (band 2), having a faste r migration in TLC analysis, was also isolated but was not reactive with PCM sera. Complete characterization of both band 1 and band 2 glycosphingolipids was undertaken by 1- and 2-D 1H- and 31P-NMR spectroscopy; electrospray mass spectrometry (ESI-MS), including low-energy CID MS/MS experiments on selected ions; exoglycosidase digestion followed by high-performance thin layer chromatography; and by GC-MS analysis of fatty acids as their methyl esters, sphingosines as their N-acetyl-O-trimethylsilyl derivatives, monosaccharides as their per-O-TMS methylglycosides, peracetylated inositols, and partially methylated alditol acetates (PMAAs). Standard 1- and 2-D 1H-NMR experiments (DQF-COSY, TOCSY, NOESY) were performed to assign as many proton resonances as possible, to establish identity and anomeric form of all monosaccharide residues, and to establish sequence and as many linkage sites as possible via interglycosidic dipolar interactions. 1-D 31P-NMR and 1H-31P correlation spectrosc opy was performed to establish the linkage positions of the phosphate groups in the lipids. ESI-MS and -MS/MS analysis (Sciex API-III) was performed on the native glycosphingolipids to confirm glycan sequence and phosphoceramide features. An aliquot (100 (g) of each lipid was permethylated by the Hakomori method, depolymerized, and derivatized to PMAAs for GC-MS analysis (Hewlett-Packard 5890 GC/5970 MSD, DB-5 column). Identification of PMAAs was made by retention times and EI fragmentation patterns compared with known standards; these confirmed both the identity and linkage form of all hexose residues in the glycans. A manuscript concerning this work has recently been published. More recently, we have begun work on glycosylinositol phosphorylceramide antigens from Aspergillus fumigatus, another mycopathogen with growing importance worldwide. A similar elucidation strategy will be used for these antigens. Preliminary results of 1-D 1H-NMR spectroscopy indicate the presence of a series of antigens similar to those already described for P. brasiliensis; however, several more complex antigens appear to be synthesized as well by A. fumigatus. These are currently under study. Finally, we have also initiated studies of the structures of antigenic monohexosylceramides of A. fumigatus, P. brasiliensis, and a variety of other mycopathogens. Similarities have been observed, with respect to both sugar and ceramide components, between the major A. fumigatus and P. brasiliensis monohexosylceramide antigens. A. fumigatus appears to be capable of synthesizing both glucosyl- and galactosylceramides, while P. brasiliensis synthesizes only glucosyl ceramides. Both exhibit ceramide structures that are not found in mammals but have been demonstrated previously in glycosphingolipids from a variety of fungi and marine invertebrates such as sponges, anemones, and starfish. Interestingly, however, the major glucosylceramides of the two forms of P. brasiliensis differ by the presence of a single unsaturation in the fatty acid component of the mycelium form. A manuscript describing this work is in preparation.